活血定眩胶囊含药血清对氧糖剥夺诱导的bEnd.3细胞铁死亡的影响  

Effect of Huoxue-Dingxuan capsule-containing serum on ferroptosis of bEnd.3 cells induced by oxygen-glucose deprivation

作　　者：王凯 宋敏[1,2] 宋志靖 李金益[1] 范凯 海云翔 WANG Kai;SONG Min;SONG Zhi-jing;LI Jin-yi;FAN Kai;HAI Yun-xiang(Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;The Affiliated hospital of Gansu University of Chinese Medicine,Lanzhou 730020,China)

机构地区：[1]甘肃中医药大学,甘肃兰州730000 [2]甘肃中医药大学附属医院,甘肃兰州730020

出　　处：《中国病理生理杂志》2022年第3期420-426,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81360554,No.81760876);甘肃省自然科学基金资助项目(No.1610RJZA069);甘肃省教育厅优秀研究生“创新之星”项目(No.2021CXZX-765);甘肃省高等学校科学研究项目(No.2017A-053)。

摘　　要：目的:探讨活血定眩胶囊(HXDX)对氧糖剥夺(OGD)诱导小鼠脑微血管内皮细胞株bEnd.3铁死亡的影响。方法:将bEnd.3细胞分为空白对照(control)组、OGD组、OGD+铁死亡抑制剂ferrostatin-1(Fer-1)组及OGD+HXDX组,control组细胞正常培养,其他组建立OGD模型,OGD+Fer-1组和OGD+HXDX组分别加入1μmol/L Fer-1和10%HXDX含药血清后OGD处理6 h。FerroOrange探针测定细胞内Fe^(2+)水平,DCFH-DA探针检测活性氧(ROS)含量,试剂盒检测超氧化物歧化酶(SOD)活性及丙二醛(MDA)和谷胱甘肽(GSH)水平,Western blot检测核因子E2相关因子2(Nrf2)、铁蛋白重链1(FTH1)、谷胱甘肽过氧化物酶4(GPX4)、长链脂酰辅酶A合成酶4(ACSL4)、环加氧酶2(COX2)和NADPH氧化酶1(NOX1)蛋白表达。结果:与control组比较,OGD组bEnd.3细胞内Fe^(2+)荧光强度与ROS含量显著增加;SOD活性降低,GSH表达下降,MDA含量显著增高(P<0.05);FTH1和GPX4表达降低,ACSL4、COX2和NOX1表达升高(P<0.05),Nrf2无显著差异。HXDX含药血清处理后,Fe^(2+)水平和ROS含量显著降低(P<0.05);SOD活性和GSH含量显著升高,MDA水平显著降低(P<0.05);Nrf2、FTH1与GPX4蛋白的表达显著增高(P<0.05),COX2和NOX1蛋白表达降低(P<0.05),ACSL4无显著差异。结论:活血定眩胶囊可抑制体外OGD诱导的bEnd.3细胞铁死亡,其机制与降低细胞内Fe^(2+)和ROS含量、减轻脂质过氧化及调控铁死亡相关蛋白表达有关。AIM:To investigate the effect of Huoxue-Dingxuan capsule(HXDX)on the ferroptosis of mouse brain microvascular endothelial cell line bEnd.3 induced by oxygen-glucose deprivation(OGD).METHODS:The bEnd.3 cells were divided into control group,OGD group,OGD+ferroptosis inhibitor ferrostatin-1(Fer-1)group and OGD+HXDX group.The cells in control group were cultured normally,while those in other groups were subjected to OGD.The cells in OGD+Fer-1 group and OGD+HXDX group were treated with 1μmol/L Fer-1 and 10%HXDX-containing serum,respectively,and treated with OGD for 6 h.FerroOrange and DCFH-DA probes were used to measure the levels of Fe^(2+)and reactive oxygen species(ROS),respectively,and the levels of superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione(GSH)were measured by commercial kits.The protein levels of nuclear factor E2-related factor 2(Nrf2),ferritin heavy chain 1(FTH1),glutathione peroxidase 4(GPX4),long-chain acyl-CoA synthase 4(AC-SL4),cyclooxygenase 2(COX2)and NADPH oxidase 1(NOX1)were detected by Western blot.RESULTS:Compared with control group,the Fe^(2+) and ROS levels were significantly increased,SOD activity and GSH expression decreased,and MDA content increased significantly in OGD group(P<0.05).The expression of FTH1 and GPX4 was decreased,while the levels of ACSL4,COX2 and NOX1 were increased(P<0.05),and there was no significant difference in Nrf2.The Fe^(2+),ROS and MDA levels were significantly decreased,while SOD activity and GSH content were significantly increased after treatment with HXDX-containing serum(P<0.05).The expression of Nrf2,FTH1 and GPX4 were significantly increased(P<0.05),while COX2 and NOX1 were decreased(P<0.05),and there was no significant difference in ACSL4.CONCLUSION:Huoxue-Dingxuan capsule inhibits OGD-induced ferroptosis of bEnd.3 cells in vitro,which is related to reducing Fe^(2+) and ROS levels,attenuating lipid peroxidation and regulating ferroptosis-related protein expression.

关 键 词：椎动脉型颈椎病 活血定眩胶囊 bEnd.3细胞 氧糖剥夺 铁死亡 

分 类 号：R743[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学]